| Objective:Previous studies reported that y-aminobutyric acid (GABA) and GABAA receptors (GABAARs) exist in human and rodent hepatocytes. The objective of my thesis study is to establish the GABAergic signaling system in the liver of rats and to investigate the role of this system in regulation of liver functions.Methods:Expressions of GAD, the rate-limiting enzyme that is involved in GABA synthesis, and GABA receptors were investigated by RT-PCR, Western Blot assays, immunofluorescence staining, and patch-clamp experiments. A rat model of ALF induced by two intraperitoneal (i.p.) injections of D-galactosamine (GalN) (0.83 g/kg body weight, at a 12-hour interval) was established, and i.p. injections of GABA, L-AG (a selective GAD inhibitor), muscimol (a selective GABAAR agonist), and baclofen (a selective GABABR agonist) were used to investigate the physiologic and pathophysiologic roles of hepatic GABA-ergic system in rats. Normal rat primary hepatocytes were used to investigate the effect of activating hepatic GABA-ergic system on the hepatotoxicity of GalN. Immunofluorescence staining showed that GAD65/67 was expressed in the cytoplasma of hepatocytes that distributed around the central vein, while GABAAR subunitsβ2 andβ3 (hereafter named as GABAAR 02/3), which are required for most functional GABAARs in neurons, were expressed on the membrane of most hepatocytes. In addition, GABABR lb was expressed on the membrane and cytoplasma of most hepatocytes. These results suggest that there is a GABAergic system in the liver.2. Consistent with above mentioned results, whole cell patch-clamp recordings in rat liver Clone 9 cells demonstrated that GABA induced a rapid inward current which was sensitive to the GABAAR antagonist picrotoxin, suggesting hepatocytes possess functional GABAAR channels (n=17 out of 56 tested cells). Treatment with GABA for 12～18 hours depolarized WITT human cholangiocarcinoma cells from -25.16 mV±2.94 mV (n=24) to -16.53 mV±1.64 mV (n=14). GABA evoked both phasic (4 out of 78 tested cells) and tonic currents (8 of 78 tested cells) on WITT cells with amplitudes of 13.2 pA±0.43 pA and 3.27 pA±0.39 pA, respectively. These results suggest that GABAAR subunits form functional GABAARs in the liver.3. Administration of GABA or L-allylglycine (L-AG) by i.p. injection for 3 days did not affect liver function in rats. Body weight gains, serum glucose level, and liver morphology were not significantly changed by GABA or L-AG compared with the controls. In addition, liver function test showed that serum total bilirubin, ALT, and AST in GABA or L-AG treated rats were similar to those of controls.4. A rat model of ALF was successfully established by i.p injection of Galactosamine (GalN). Boday weight gains in rats from GalN+saline group were decreased to 52.28%±25.95% of those in control group (n=3, P<0.05). HE staining showed disorganized liver plate and sinusoid architecture and extensive areas of necrosis in ALF rats. PAS staining showed a dramatically weak staining in liver tissues from the ALF group. In consistent with this result, serum glucose was dramaticly lower in rats from GalN+saline group in comparison to that of controls (77.43%±5.71% of control, P<0.05, n=3). Liver function test showed that serum total bilirubin, ALT and AST were increased comparing to those of controls (34.40μM±9.43μM vs. 1.30 μM±0.40μM,129.33 IU/L±45.10IU/L vs.55.00 IU/L±6.26 IU/L, and 253.00 IU/L±51.02 IU/L vs.112.38 IU/L±11.73 IU/L, respectively, P< 0.05).5. Administration of GABA (1.5 mg/kg body weight for 5 days) by i.p injection ameliorated GalN induced ALF. Body weight gains in rats from GalN+GABA group were increased compared with those in GalN+saline group (77.45%±7.93% vs. 52.28%±25.95%, P<0.05). Improvement of liver histological findings was detected by HE staining in GalN+GABA group. PAS staining showed more glycogen deposition in liver tissues of GalN+GABA group, which was consistent with the result that increased blood glucose levels were observed in GalN+GABA group (86.13%±4.35% vs.77.43%±5.71%, P<0.05). TUNEL staining showed that GalN induced apoptosis of hepatocytes was reversed by GABA treatment. In addition, serum total bilirubin, ALT and AST levels were significantly lower in GalN +GABA group in comparison to those in GalN+saline group (2.26μM±0.79μM vs. 34.40μM±9.43μM,57.86 IU/L±6.84 IU/L vs.129.33 IU/L±45.10 IU/L, and 155.29 IU/L±29.53 IU/L vs.253.00 IU/L±51.02 IU/L, respectively, P<0.05). The effects of GABAA R agonist muscimol but not GABABR agonist baclofen on the outcomes of GalN induced ALF were similar to those of GABA.6. Increased expression of GAD65/67, decreased GABAARβ3 subunit, and decreased expression of glutamine synthetase (GS), which synthesizes glutamine from glutamate and ammonia, in ALF rat livers were all reversed by muscimol but not by baclofen. In addition, the expressions of several molecules associated with the PI3 Kinase signaling pathway including PI3K, phospho-Akt, and phospho-GSK-3βwere decreased in ALF liver tissues, but they were all reversed by the selective GABAAR agonist muscimol.7. In CA1 area of the hippocampus from GalN treated rats, most neurons showed shrink and irregular morphologies comparing to those of controls (the diameter of nuclear in control rats is 17.24μm±0.64μm, n=12, while that in ALF rats decreased to 11.58μm±0.66μm, n=7,P<0.05), suggesting damages to these neurons. In contrast, treatment of GABAAR agonist muscimol but not GABABR baclofen restored these shrinked neurons to normal circular shapes (the diameter of nuclear in ALF+MUS group is 16.41μm±0.63μm, n=8, P<0.05 vs. ALF). Neither GAD65/67 nor GS expressions in the cortex of ALF rats were changed in comparison with the controls. Neither muscimol nor baclofen affected the expression of GAD65/67 or GS in the cortex.8. In vitro, the damaged morphology, decreased cell viability, and increased number of apoptotic cells induced by GalN were all reversed by GABA or muscimol treatment in rat primary hepatocytes.Conclusions:There is a functional GABA-ergic system in the rat liver, which may regulate the function of liver and bile duct through autocrine and paracrine mechanisms. Activation of this hepatic GABAergic system by exogenous GABA or muscimol improves the outcome of GalN induced ALF in rats. The GABAARs-PI3K-Akt-GSK3βpathway is likely involved in the protecting role of hepatic GABA system. |
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